| Objective:The regulation of cancer cells, especially cancer stem cells (CSCs) by tumor microenviroment play a key role in the progression, metastasis and prognosis of cancer, so it is important to clarify the regulatory mechanism. CD4+CD25+regulatory T cells (Tregs) in the tumor microenviroment are thought to be one of the major suppressors of anti-tumor immunity, but its direct action on CSCs has not been reported. This study was focused on the regulatory effects of Tregs on CSCs in breast cancer and investigated the possible role of epithelial-mesenchymal transition (EMT) induced by TGF-β1during this process.Methods:1. The expression of Foxp3and ALDH1was assayed by immunohistochemisty in paraffin-embedded tumor specimens abtained from104cases of invasive breast cancer patients who experienced radical mastectomy in Tianjin Medical University Cancer Hospital. The result was analysed according to the clinical and pathological characters, and the correlation between Foxp3+Tregs and ALDH1+CSCs like cells was further investigated.2. CD4+CD25+Tregs isolated from the peripheral blood of breat cancer patients were incubated with breast cancer cell line BT474and MCF-7. Then the EMT related biomarkers and transcription factors of cancer cells were assayed before and after incubation with Tregs. The ability of migration and invasion was detected either. Further more, the changes of ALDH1(a biomarker of CSCs) was detected by flow cytometry, real-time PCR, and western blot. The mamosphere forming ability, self-renewal capacity in vitro, and the tumorigenicity in vivo also assayed.3. The neutralization antibody of TGF-β1and the inhibitor of TGF-β receptor were used to investigate the role of TGF-β1in the process of EMT and acquisition of stemness. What’s more, an isolation culture system in which Tregs and BT474cells were separated by a membrane was established to explore the possible regulatory mechanism of Tregs on CSCs property. Results:1.The ALDH1positive cells were seen in38.5%of breast cancers, and the number was increased in poorly differentiated tumors, estrogen and progesterone receptor-negative tumors and triple negative breast cancers (P=0.019,0.028,0.046and0.008, respectively). Poor differentiation and lymph node metastasis was related with more Foxp3-positive Tregs (P=0.001and0.025, respectively). There was a positive correlation between the number of ALDH1+CSCs like cells and Foxp3+Tregs (r=0.215, P=0.029).2. After incubation with Tregs, the expression of mesenchymal biomarkers was elevated in BT474and MCF-7cells, but the expression of the epithelial biomarker E-cadherin was decreased in MCF-7cells. Meanwhile, the expression of EMT related transcription factor Slug in both cell lines was increased after incubation, accompanied by the elevation of migratory and invasive capacity of tumor cells. Compared to the control group, the subpopulation of ALDEFLUOR+CSCs like cells in BT474, the mamosphere forming ability, the self-renewal capacity, and the tumorigenicity was enhanced in the coculture group.3. The EMT process and sternness induced by Tregs can be blocked by the inhibition of TGF-β1signal pathway. The concentration of TGF-β1in the supernatant was mildly elevated in the coculture group compared to the control group (P=0.047), while an apparent TGF-β1expression was detected on the surface of Tregs by immunofluorescence. The proportions of ALDEFLUOR+cells in BT474were45.2%,73.4%,50.1%, and50.6%in the control group, coculture group, isolation culture group, and supernatant of Tregs culture group, respectively.Conclusion:1. The Foxp3+Tregs and ALDH1+CSCs like cells can be seen in breast cancers, and there was a positive correlation between these two kinds of cells.2. The EMT process, the migratory and invasive capacity, and the sternness of breast cancer cells can be enhanced by Tregs.3. Tregs induced the EMT process and the stemness of breast cancer cells in a TGF-β1dependent manner, and mainly mediated by cell surface bound TGF-β1. |
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